The myosin heavy chain (HC) Beta is a major contractile protein of the cardiac and slow skeletal muscles. We have recently shown that the transcription of the rabbit myosin HC Beta gene is regulated through multiple cis-acting elements, two of which are essential for conferring muscle specificity on the heterologous promoter. One element interacts with muscle-specific (A1) as well as ubiquitous factors (A2) and another element interacts only with a ubiquitous factor (B). These nuclear factors seem to differ from other well-characterized factors, such as the MyoD family or general transcription factors. The goal of this proposal is to elucidate the structure and function of these nuclear factors involved in muscle-specific expression of myosin HC Beta gene in order to understand the mechanisms by which these factors contribute to the tissue-specific activation of this gene. First, we will attempt to isolate the cDNAs encoding these factors by screening a lambda gt11 muscle expression cDNA library with oligonucleotides containing the factor binding sites. At the same time, we will examine whether the A1 and A2 factors are related to the SV40 transcriptional enhancer factor 1 (TEF-1) because the DNA sequence of the TEF-1 binding site is nearly identical to the A1 and A2 factor binding sequence. We will use antisera against TEF1 to investigate whether the A1 and A2 factors are TEF-1 related proteins. If so, we will screen a lambda GT11 muscle cDNA library with either antisera to with TEF-1 or TEF-1 cDNA. If above approaches are not successful, we will attempt to purify A1, A2 and B factors from a large scale preparation of nuclear extracts from cultured myotube by sequence-specific DNA-affinity columns and other conventional column chromatography. The purified proteins are subjected to microsequencing and the oligonucleotides corresponding to the peptide sequences will be used as cDNA screening probes. If peptide sequences cannot be obtained, we will raise specific antibodies against the purified factors and use the antibodies to screen the cDNA library. Once the cDNAs are isolated, we will examine the ability of these factors to activate the myosin HC Beta gene promoter by DNA transfection or by in vitro transcription. Finally, we will examine the physiological roles of these factors during myogenesis by developing the stably transfected myogenic cell lines that conditionally express anti-sense RNAs against these factors. These studies may reveal the mechanisms by which both muscle-specific and ubiquitous factors are needed for muscle-specific activation of the contractile protein genes.